Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remain the most important features when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicate vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included and is compatible with <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i> applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells and iPSCs in vitro, as well as <i>in vivo</i> transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we identified also novel previously unknown tropisms for some AAV variants.

Lung gene therapy requires efficient transduction of slow-replicating epithelia and stable expression of delivered transgenes in the respiratory tract. Lentiviral vectors (LV) have the ideal coding, expression, and transducing capacity required for gene therapy. A modified envelope glycoprotein from the Jaagsiekte Sheep Retrovirus, termed Jenv, is well suited for LV-mediated lung gene therapy due to its inherent lung tropism. Here, two novel Jenv-pseudotyped LVs that effectively transduce lung tissue and yield titers similar to the gold standard, vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped LVs, were generated. As concentration efficiency of LVs was found to depend on envelope pseudotype, a large-scale production method tailored for Jenv pseudotyped LVs was developed and the most appropriate method of concentration determined. In contrast to VSVg and Ebola virus glycoprotein-pseudotyped LVs, ultracentrifugation through a sucrose cushion drastically reduced the yield of Jenv LVs, whereas polyethylene glycol precipitation and tangential flow filtration (TFF) proved to be more suitable methods for concentrating Jenv LVs. Importantly, pressure during TFF was found to be crucial for increasing LV recovery. Finally, a unique mouse model was developed to test the suitability of these novel Jenv pseudotyped LVs for use in lung gene therapy applications.

The pro-renin receptor (PRR), as an important novel component of the renin-angiotensin system, has multifunction yet not completely known. In this study, we aimed to explore the effect of PRR on the formation of Ang II-induced abdominal aortic aneurysm (AAA) in apolipoprotein E-knockout mice. We used Ang II (1.44 mg/kg/day) infusion to induce AAA, then mice received different treatment with saline, telmisartan, no treatment, Ad-EGFP, Ad-PRR, or Ad-PRR plus telmisartan. The incidence of AAA was 35%, 60%, 65%, 90%, and 55% in the Telmisartan, Vehicle, Ad-EGFP, Ad-PRR, and Ad- PRR+Telmisartan groups, respectively. Compared with the Vehicle and Ad-EGFP groups, PRR overexpression markedly increased macrophage infiltration; levels of proinflammatory cytokines, including MCP-1 and TNF-α; the expression and activity of MMP2 and MMP9; NOX2 and NOX4 protein and mRNA expression; NADPH oxidase activity; ERK and P38MAPK expression; but decreased smooth muscle cells content in AAA. However, telmisartan reversed the adverse effects of PRR. In addition, ERK inhibitor PD98059 eliminated the acceleration of Ang II-induced AAA formation by PRR and co-administration of telmisartan and PD98059 further abolished the adverse effects of PRR on Ang II-induced AAA formation. Thus, PRR plays an important role in the pathological development of AAA via both Ang II-dependent and -independent activation of ERK pathways and inhibition of PRR activation may be a promising approach to the treatment of AAA.

High-risk human papillomavirus (HPV) E6 and E7 genes display vital oncogenic properties in cervical cancer. Eliminating HPV driver gene or lose-of-function by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a promising treatment for the HPV-associated cancer. Thus, the present study designed a CRISPR/Cas9 system to target the E6 and E7 genes at once, to detect whether it have efficacy in vitro and in vivo. Meanwhile, CRISPR/Cas9 system were measured following transfection with Liposomes but virus. Cervical cancer lines (HeLa and SiHa) were used in this study. Sanger sequencing confirmed that the single CRISPR/Cas9 vector [termed E6E7-knockout (KO)] containing guide gRNAs could targeting both HPV 18 E6 and E7 genes in vitro. In addition, Double-targeting E6 and E7 increased p53 protein expression significantly while compared with E6 or E7 targeting respectively. Mice with xenografts were divided into 4 groups: 3 doses of experimental groups (20 µg, 40 µg and 60 µg) and 1 control group. The E6E7-KO via Liposome delivery was injected into tumours. Tumour growth was measured and protein expression was observed via immunohistochemistry. The toxic side-effects in vivo were also evaluated. E6E7-KO induced cell apoptosis and inhibited cell proliferation markedly in vitro. E6E7-KO downregulated the mRNA and protein expression of E6 and E7 while p53 and p21 protein levels were upregulated accordingly. Notably, E6E7-KO delivery by Liposome exhibited an effect in vivo. Tumour growth was inhibited in the E6E7-KO groups, which was accompanied by decreased E6/E7 protein expression and increased p53/p21 protein expression, especially the level of p53 protein expression. Therefore, E6E7-KO could have synergy efficient by p53 pathway. Furthermore, local injection with CRISPR/Cas9 by non-viral delivery may be regarded as a potential therapy for cervical cancer in the future.

Renal fibrosis leads to end-stage renal disease, but anti-fibrotic drugs are difficult to develop. Chronic kidney disease often results in muscle wasting and thereby increases morbidity and mortality. In this work, adeno-associated virus (AAV)-mediated overexpressing miR-29a was hypothesized to counteract renal fibrosis and muscle wasting via muscle-kidney crosstalk in unilateral ureteral obstruction (UUO) mice. miR-29a level was downregulated in the kidney and skeletal muscle of UUO mice. The secretion of exosome-encapsulated miR-29a increased in cultured skeletal muscle satellite cells and HEK293 renal cells after stimulation with serum from UUO mice. This result was confirmed by qPCR and microRNA deep sequencing in the serum exosomes of mice with obstructed ureters. A recombinant AAV-miR-29a was generated to overexpress miR-29a and injected into the tibialis anterior muscle of the mice 2 weeks before UUO surgery. AAV-miR-29a abrogated the UUO-induced upregulation of YY1 and myostatin in skeletal muscles. Renal fibrosis was also partially improved in the UUO mice with intramuscular AAV-miR-29a transduction. AAV-miR-29a overexpression reversed the increase in TGF-β, fibronectin, alpha-smooth muscle actin, and collagen 1A1 and 4A1 levels in the kidney of UUO mice. AAV-GFP was applied to trace the AAV route in vivo, and fluorescence was significantly visible in the injected/ un-injected muscles and in the kidneys. In conclusion, intramuscular AAV-miR-29a injection attenuates muscle wasting and ameliorates renal fibrosis by downregulating several fibrotic-related proteins in UUO mice.

Gene therapy with adeno-associated viral (AAV) vectors has reached the clinical stage for many inherited and acquired diseases. However, due to a cargo capacity limited to <5 kb, AAV-mediated treatment of diseases that require transfer of larger genes still appears elusive. This is a major drawback of a platform that has otherwise been repeatedly found to be safe and effective. Thus, great efforts have been directed toward the identification of strategies to overcome this limitation. Among the most studied approaches is the use of dual vectors, in which a transgene is split across two separate AAV vectors. Mechanisms acting at either the DNA, pre-mRNA, or protein levels have been explored to restore full-length transgene expression in infected cells. Here, we will review them as well as additional strategies developed to deliver large genes with AAV. We discuss the pros and cons of these strategies and the aspects that still need to be addressed.

Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the AAV genome is replaced with a gene cassette of interest, leaving only the 145 bp inverted terminal repeat sequences. These cis-elements primarily from AAV serotype 2 are required for genome rescue, replication, packaging, and vector persistence. Previous work from our lab and others have demonstrated that the AAV ITR2 sequence has inherent transcriptional activity, which may confound intended transgene expression in therapeutic applications. Currently, AAV capsids are extensively study for vector contribution, however, a comprehensive analysis of ITR promoter activity of various AAV serotypes has not been described to date. Here, the transcriptional activity of AAV ITRs from different serotypes (1-4, 6 and 7) was compared in numerous cell lines and a mouse model. Under the conditions used here, all ITRs tested were capable of promoting transgene expression in vitro and in vivo. However, we observed 3 classes of AAV ITR expression in vitro. Class I ITRs (AAV2 and 3) generated the highest level, while class II (AAV 4) intermediate levels, and class III (AAV1 and 6) had the lowest. These expression levels were consistent across multiple cell lines. Only ITR7 demonstrated cell-type dependent transcriptional activity. In vivo, all classes had promoter activity. Next generation sequencing revealed multiple transcriptional start sites that originated from the ITR sequence, with most arising from within the RBE. The collective results demonstrate that the serotype ITR sequence may have multiple levels of influences on transgene expression cassettes independent of promoter selection.

The NGVB program has been highly accessed by gene therapy investigators. The Reagent Repository has distributed over 1,000 reagents to 397 investigators. The Pharmacology/Toxicology Archive contains over 36,000 specimens from a variety of AAV, Adenoviral and other Pharmacology/Toxicology studies. NGVB also maintains a searchable database of Gene Therapy Pharmacology/Toxicology studies to promote data sharing. NGVB has provided FDA mandated replication competent virus testing for over 70 clinical trials. From 2008 through 2018, there have been 114 publications acknowledging the NGVB. It is unlikely that any other NIH funded program has served as many gene therapy investigators as the NGVB.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated rAAV2tYF-GRK1-hRPGRco, to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (hRPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid variant with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a toxicity and efficacy study of this vector administered by subretinal injection in the naturally occurring RPGR mutant (XLPRA2) dog model. Sixteen RPGR mutant dogs divided into 4 groups of 3 to 5 animals each received either a subretinal injection of 0.07 mL of AGTC-501 at low (1.2 x 1011 vector genome [vg]/mL), mid (6 x 1011 vg/mL) or high dose (3 x 1012 vg/mL), or of vehicle-control in the right eye at early stage disease. The left eye remained untreated. Subretinal injections were well tolerated and were not associated with systemic toxicity. Electroretinography (ERG), in vivo retinal imaging, and histological analysis showed rescue of photoreceptor function and structure in absence of ocular toxicity in the low- and mid-dose treatment groups when compared to the vehicle-treated group. The high-dose group showed evidence of both photoreceptor rescue and posterior segment toxicity. These results support the use of AGTC-501 in clinical studies with patients affected with XLRP caused by RPGR mutations and define the no-observed-adverse-effect-level (NOAEL) at 6 x 1011 vg/mL).

Objective Coronary artery disease is a major cause of death and disability worldwide. New therapies are needed for patients who do not benefit or are not suitable for current treatments. Angiogenic gene therapy using vascular endothelial growth factors (VEGFs) has shown potential in preclinical trials. However, undesired side-effects, such as increased permeability, limit their therapeutic potential. The aim of this study was to investigate if adenoviral gene transfer of a VEGF receptor 2 (VEGFR-2) ligand Gremlin, given simultaneously with VEGF-A, could modulate VEGFR-2 mediated increase in permeability without impairing the angiogenic effect of VEGF-A gene therapy. Methods Gene transfers were done in pigs (n=22) using endocardial injections with an endovascular injection catheter. Animals were divided in three groups receiving adenoviral (Ad) VEGF-A (n=10), Gremlin (n=6) or VEGF-A + Gremlin (n=6) gene therapy. Animals were sacrificed and samples collected six days later for histological, safety and permeability analyses. Results The mean capillary area was significantly increased in both treatment groups with AdVEGF-A when compared to the AdGremlin group. Also, the capillary area was significantly larger in AdVEGF-A group without AdGremlin. No significant differences in tissue permeability were observed using modified Miles assay between AdVEGF-A and AdVEGF-A + AdGremlin groups. However, cardiac tamponade and sudden cardiac deaths were observed only in the AdVEGF-A group. Conclusion AdVEGF-A induces strong angiogenesis in porcine myocardium. Our results suggest that AdGremlin can limit the side effects of AdVEGF-A therapy, even though no direct effect on tissue permeability could be demonstrated. This could enable the use of larger AdVEGF-A doses to increase the treatment area and angiogenic effects without adverse side effects.

Adeno-associated viral vector (AAV) is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for the treatment of various genetic disorders, including muscular dystrophies. These dystrophies represent an heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency via direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different rAAV serotypes is therefore needed. Here, we investigated the transduction efficiency of rAAV serotypes 1 to 10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of beta-galactosidase activity on muscle cryo-sections demonstrated that rAAV1, 7, 8, 9 and 10 were more efficient than the others with rAAV9 being the most efficient in mouse. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.